Peptides and derivatives thereof showing cell attachment, spreading and detachment activity

ABSTRACT

The present invention relates to peptides and derivatives thereof showing cell attachment, spreading and detachment activity. Particularly, the present invention relates to the peptide NKDIL and EPDIM and derivatives thereof which promote the cell attachment activity through interaction with a α 3β 1 integrin as a functional cell receptor and include aspartic acid and isoleucine essential for cell attachment and detachment activity. The peptides and derivatives thereof in the present invention can be used for developing a study of cell attachment activity mediated through various extracellular matrix protein containing α ig-h3, wound healing, tissue regeneration and metastasis inhibition.

CONTINUING DATA

[0001] The present application is a U.S. national phase applicationunder 35 U.S.C. §371, of PCT/KR00/01413, filed Dec. 6, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to peptides and derivatives thereofshowing cell attachment, spreading and detachment activity.Particularly, the present invention relates to the peptide NKDIL andEPDIM and derivatives thereof which promote the cell attachment activitythrough interaction with α3β1 integrin as a functional cell receptor andinclude aspartic acid and isoleucine essential for cell attachment anddetachment activity.

BACKGROUND OF THE INVENTION

[0003] βig-h3 is an extracellular matrix protein whose expression isinduced in various cell lines, including human melanoma cells, mammaryephithelial cells, keratinocytes, and lung fibroblasts, followingsignaling by active TGF-β. The βig-h3 gene was first isolated bydifferential hybridization screening of a cDNA library made from a humanlung adenocarcima cell line that had been treated with TGF-β. βig-h3gene encodes a 683-amino acid protein that is highly conserved betweenspecies. It contains an N-terminal secretory signal peptide and anArg-Gly-Asp (RGD) motif at the C-terminus. The RGD motif, whichmodulates cell adhesion, is found in many extracellular matrix proteinsand serves as a ligand recognition sequence for several integrins.

[0004] Because the expression of βig-h3 gene is increased by TGF-β invarious cell lines and the gene is induced in various cell lines whoseproliferation rate is controlled by TGF-β, βig-h3 is believed to beinvolved in mediating some of the signaling pathways of TGF-β. Incontrast, βig-h3 expression is reported to be reduced in the fibroblastscultured from the skin lesions afflicted with localized hyperostosis ofmelorheostosis, some tumor cells, and dexamethasone-treated stem cells.Accordingly, βig-h3 plays an important role in the morphogenesis andinteractions with cells and other extracellular matrix proteins invarious tissues.

[0005] Additionally, βig-h3 is known to mediate cell attachment anddetachment, serving as a cell adhesion molecule. Purified βig-h3 proteinis found to promote the attachment and spreading of skin fibroblastswhile inhibiting the adhesion of A549, HeLa and Wi-38 cells inserum-free media. Particularly, βig-h3 is known to have inhibitoryactivity against tumor cell growth, and colony formation. In fact, itwas reported that βig-h3 remarkably suppressed the growth of CHO(Chinese hamster ovary) cells in nude mice. Furthermore, a wound healingmethod was developed on the basis of the finding that application of apharmaceutically effective amount of βig-h3 to wounds makes cells,especially fibroblasts, spread over and adhere to the wounded site.Consequently, βig-h3, a cell adhesion molecule induced by TGF-β invarious cell lines, plays a very important roles in cell growth, celldifferentiation, wound healing, morphogenesis and cell adhesion.

[0006] βig-h3 contains four 140 amino acid repeats with internalhomology. The internally repeated domains have highly conservedsequences found in secretory proteins or membrane proteins of variousspecies, including mammals, insects, sea urchin, plants, yeast andbacteria. Proteins such as periostin, fasciclin I, sea urchin HLC-2,algal-CAM and mycobacterium MPB70 are examples containing the conservedsequences. The homology domain conserved in these proteins (hereinafterreferred to as “fas-1”) consists of about 110 to 140 amino acids withtwo highly conserved branches of H1 and H2 which have about 10 aminoacids each. Four fas-1 domains are found in βig-h3, periostin, andfasciclin I, two fas-1 domains in HLC-2, and only one fas-1 domain inMPB70. Although the functions of the proteins are not elucidatedclearly, some of them are known to act as cell adhesion molecules. Forinstance, βig-h3, periostin, and fasciclin 1 are reported to mediate theadhesion of fibroblasts, osteoblasts, and nerve cells, respectively.Also, it is disclosed that the algal-CAM is a cell adhesion moleculepresent in embryos of algae Volvox.

[0007] The cell attachment activity of βig-h3 was found first in humandermal fibroblasts and then in chondrocytes, peritoneal fibroblasts andhuman MRC5 fibroblast. At first, it was believed that the cellattachment activity of βig-h3 would be mediated by the C-terminal RGDmotif. However, some research results revealed that the RGD motif is notnecessary for promoting the spread of chondrocytes and that the maturesoluble βig-h3 whose RGD motif is deleted by carboxyl-terminusprocessing is able to inhibit cell adhesion, leading to the conclusionthat the RGD motif of βig-h3 is dispensable for mediating the cellattachment activity of βig-h3. In addition, it has been recentlyreported that βig-h3 promotes the spread of fibroblasts via integrinα1β1 whereas the RGD motif of βig-h3 is not necessary forβig-h3-mediated cell spreading. Further, the conserved peptides H1 andH2 of βig-h3 do not inhibit βig-h3-mediated cell adhesion, so that theconserved peptides are not effective for βig-h3-mediated cellattachment. These results, taken together, indicate that amino acidsindispensable for the cell attachment activity of βig-h3 exist inregions other than H1 and H2. A computer analysis of the homology amongfas-1 domains of other proteins as well as between repeated fas-1domains of βig-h3 discloses the existence of several highly conservedamino acid sequences in addition to H1 and H2 peptides, suggesting theposibility of the involvement of the conserved amino acid sequences inthe cell attachment activity.

SUMMARY OF THE INVENTION

[0008] Leading to the present invention, the intensive and thoroughresearch on conserved motifs responsible for the cell attachment anddetachment activity, conducted by the present inventors, resulted in thefinding that aspartic acid and isoleucine at positions near the H2region within each of the 2^(nd) and 4^(th) fas-1 domains of βig-h3 arevery highly conserved and are identified as a functionally essentialunit for mediating cell adhesion through α3β1 integrin.

[0009] Therefore, it is an object of the present invention to providepeptides and their derivatives, which contain conserved amino acidsequences essential for cell attachment, spreading and detachmentactivity.

[0010] It is another object of the present invention to provide apharmaceutical compositions for use in wound healing, tissueregeneration and cancer metastasis resistance.

[0011] In accordance with one embodiment of the present invention, thereis provided a peptide having cell attachment, spreading and detachmentactivity, comprising an amino acid sequence represented by the followingone-letter symbols: XXDIX wherein X is any of the twenty common aminoacids, D stands for aspartic acid, and I stands for isoleucine, andderivatives thereof.

[0012] In accordance with another aspect of the present invention, thereis provided a pharmaceutical composition, comprising the above peptideor its derivative as a pharmaceutically active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1a is a schematic diagram showing recombinant proteinsderived from fas-1 domains of βig-h3.

[0014]FIG. 1b is a photograph showing SDS-PAGE results of therecombinant proteins derived from fas-1 domains of βig-h3.

[0015]FIG. 2 is a histogram showing cell attachment activity of therecombinant proteins derived from fas-1 domains of βig-h3.

[0016]FIG. 3 is a histogram showing the inhibitory activity ofanti-integrin antibody against the cell attachment activity of therecombinant proteins derived from fas-1 domains of βig-h3.

[0017]FIG. 4 shows amino acid sequences of various matrix proteinscontaining fas-1 domains.

[0018]FIG. 5a is a schematic diagram showing substitution mutantsderived form the 4^(th) fas-1 domain of βig-h3.

[0019]FIG. 5b is a photograph showing 15% polyacrylamide gelelectrophoresis results of substitution mutants derived from the 4^(th)fas-1 domain of βig-h3.

[0020]FIG. 6 is a histogram showing the cell attachment activity ofsubstitution mutants derived from the 4^(th) fas-1 domain of βig-h3.

[0021]FIG. 7 shows amino acid sequences of the synthetic peptides of thepresent invention.

[0022]FIG. 8a is a histogram showing the inhibitory activity of thesynthetic peptides against HCE cell adhesion.

[0023]FIG. 8b is a histogram showing inhibition of HCE cell adhesion toβig-h3 D-II or βig-h3 D-IV proteins by the synthetic βig-h3 peptides.

[0024]FIG. 9a is a curve showing HCE cell attachment activity accordingto the synthetic peptides of the present invention.

[0025]FIG. 9b is a histogram showing the inhibition of anti-integrinantibodies against HCE cell adhesion to the synthetic peptides EPDIM andNKDIL.

[0026]FIG. 10a is a histogram showing effects of the synthetic peptidesof the present invention on HCE cell adhesion to several extracellularmatrix proteins.

[0027]FIG. 10b is a curve showing dose-dependent inhibition of HCE celladhesion surfaced coated with extracellular matrix proteins in thepresence of various concentrations of EPDIM.

DETAILED DESCRIPTION OF THE INVENTION

[0028] In the present invention, the amino acid sequence of βig-h3,known to mediate cell attachment activity, is utilized to preparepeptides comprising conserved sequences essential to cell attachment andspreading activity, based on the finding that not only βig-h3 comprising4 fas-1 domains, but also either the 2^(nd) domain or the 4^(th) domainalone can mediate the cell attachment activity.

[0029] In more detail, four different truncated DNA fragments of theβig-h3 gene, that is, βig-h3 D-I, βig-h3 D-II, βig-h3 D-III, and βig-h3D-IV, which encode 1^(st) to 4^(th) internal repeat domains,respectively, were synthesized as shown in FIG. 1a. A quantitativemeasurement which shows the cell attachment and spreading activity ofthe resulting four recombinant proteins demonstrates that only eitherthe 2^(nd) or 4^(th) domain of the four fas-1 domains can mediate thecell attachment and spreading activity of βig-h3, as shown in FIG. 2,indicating the existence of amino acids essential to the mediation ofthe cell attachment and spreading in the two domains. In contrast, the1^(st) domain of βig-h3 shows intermediate cell attachment and spreadingactivity while no cell attachment activity is found in the 3^(rd)domain.

[0030] Also, in accordance with the present invention, α3β1 integrin isidentified as the functional receptor for the 2^(nd) and 4^(th) domainsof βig-h3.

[0031] Using the truncated proteins comprising the 2^(nd) and 4^(th)domains of βig-h3, each of which shows cell attachment, spreading anddetachment activity, the present inventors examined the receptors ofβig-h3. The 2^(nd) or 4^(th) fas-1 domain-mediated cell attachmentactivity was almost completely suppressed by antibodies against α3 andβ1 integrin subunits, as shown in FIG. 3. These results mean that bothof the 2^(nd) and 4^(th) fas-1 domain bind to the functional receptorα3β1 integrin to mediate the cell attachment activity, and have aminoacids essential to mediate cell adhesion activity.

[0032] The present invention also provides peptides which comprise thecharacteristic conserved amino acid sequence Asp-Ile essential to thecell attachment, spreading and detachment activity of βig-h3 and thusshowing the same activity as that of the 2^(nd) and 4^(th) domains.

[0033] To find out amino acid sequence responsible for cell adhesion inthe 2^(nd) and 4^(th) fas-1 domains of βig-h3, which shows cellattachment, spreading and detachment activity independently, amino acidsequence alignment was done not only among the internally repeated fas-1domains of βig-h3, but also among other proteins containing fas-1domains. After that, the present inventors found out that two aminoacids, aspartic acid and isoleucine, near the H2 region are highlyconserved between various proteins, as shown in FIG. 4.

[0034] Next, the indispensability of the amino acid sequence asparticacid and isoleucine to the cell attachment activity remains to beconfirmed. In this regard, the truncated protein containing the 4^(th)fas-1 domain of βig-h3 was mutated by the substitution of proline,aspartic acid and isoleucine with serine, alanine and serine,respectively, as shown in FIG. 5a. Almost complete suppression of the4^(th) fas-1 domain-mediated cell attachment activity was observed inthe mutant proteins in which aspartic acid and isoleucine weresubstituted, confirming that the amino acid sequence aspartic acid andisoleucine is very important in mediating the cell attachment activityof βig-h3.

[0035] In addition, aspartic acid and isoleucine are both conserved inthe 2^(nd) and 4^(th) fas-1 domains of βig-h3, which are of high cellattachment activity, while only aspartic acid is conserved in the 1^(st)fas-1 domain 1, which shows intermediate cell attachment activity. Asfor the 3^(rd) fas-1 domain which shows no cell attachment activity, ithas neither of the two amino acids. Those facts are further evidenceshowing that aspartic acid and isoleucine are indispensable to mediatecell attachment and spreading activity.

[0036] With the confirmation of the indispensability of aspartic acidand isoleucine for cell attachment activity, three peptides containingthe two amino acids were synthesized from fas-1 domains I, II and IV ofβig-h3, respectively. These peptides have the conserved sequences of thefas-1 domains I, II and IV of βig-h3. That is, the peptides are designedto have KADHH (a.a. 219-223) of SEQ. ID. NO. 1, NKDIL (a.a. 354-358) ofSEQ. ID. NO. 2, and EPDIM (a.a. 615-619) of SEQ. ID. NO. 3, as shown inFIG. 7.

[0037] As a matter of course, these synthetic peptides derived from thefas-1 domains of βig-h3 were examined for cell attachment and detachmentactivity. The 2^(nd) fas-1 domain-derived synthetic peptide NKDIL andthe 4^(th) fas-1 domain-derived synthetic peptide EPDIM were measured toshow very excellent cell detachment effect with significant suppressionof the cell attachment activity, as seen in FIG. 8a. On the other hand,relatively very low cell detachment effects were observed in the 1^(st)fas-1 domain-derived synthetic peptide KADHH owing to its weaksuppression of cell adhesion, and also the control peptide DEMPI failedto show, owing to its deficiency in cell adhesion activity. Thesuppression effects against the cell attachment activity obtained whenusing the 2^(nd) and 4^(th) fas-1 domains as substrates were almost thesame as those obtained when using βig-h3 as a substrate, as shown inFIG. 8b.

[0038] As shown in FIG. 9a, the synthetic peptides NKDIL and EPDIM arecapable of mediating cell adhesion in dose-dependent manners. Moreover,the α3β1 integrin, known as a surface receptor of βig-h3, was revealedto act as a receptor in the synthetic peptides EPDIM and NKDIL-mediatedcell adhesion, as seen in FIG. 9b. For these reasons, NKDIL and EPDIMappeared to specifically compete with α3β1-interacting molecules, asshown in FIG. 10a.

[0039] In the present invention, as described above, there are providedthe peptides, NKDIL and EPDIM, which contain aspartic acid andisoleucine essential to the cell attachment and detachment activity, andare sufficient to induce cell adhesion through α3β1 integrin, which isthe functional receptor for βig-h3. These peptide sequences are derivedfrom the conserved sequence of the fas-1 domains II and IV, whichindependently induces cell adhesion. The peptides of the presentinvention can be used usefully for studying the cell attachment activitymediated through various extracellular matrix proteins, includingβig-h3, and developing cell attachment, spreading anddetachment-promoting peptides.

[0040] In accordance with a further aspect of the present invention,there are provided pharmaceutical compositions comprising the peptidesshowing cell attachment and detachment activity or their derivatives aseffective ingredients conferring wound healing and tissue regenerationability and resistance to cancer metastasis.

[0041] Administrable via oral or parenteral routes, the peptides ortheir derivatives may be used with ordinary medicine forms. That is, thepeptides or their derivatives can be formulated into various dosageforms for parenteral administration. For formulation, pharmaceuticallyacceptable diluents, expedients and/or carriers may be used, includingfillers, thickeners, binders, wetting agents, disintegrants,surfactants, etc. Dosage forms for parenteral administration includesterile aqueous solutions, non-aqueous solvents, suspensions, emulsions,freeze-dried agents, suppositories, etc. For formulation of non-aqueoussolvents and suspensions, vegetable oils, such as propylene glycol andpolyethylene glycol, or injectable esters such as ethyl oleate, may beused. As bases for suppositories, Witepsol, macrogol, Tween 61, cocoabutter, laurinic acid, and glycerogelatine are useful.

[0042] In addition, the peptides and their derivatives may be used incombination with pharmaceutically acceptable carriers such asbiologically active saline or organic solvents. Also, carbohydrates suchas glucose, sucrose, and dextran, antioxidants such as ascorbic acid andglutathion, chelating agents, low molecular weight proteins, or otherstabilizers may be employed to increase the stability or absorption ofthe peptides.

[0043] The total effective amount of the peptides may be administered asa dosage form of a bolus or may be administered by infusion in a singledose when a relatively short period of administration is desired.Alternatively, the administration of the peptides may follow amultiple-dose manner according to a fractionated treatment protocol.Depending on the ages, body conditions and body weights of the patientsas well as administration routes and treatment number, thepharmaceutically effective dosage of the peptides of the presentinvention may be varied, and can be easily determined by those skilledin the art.

[0044] Because the pharmaceutical compositions comprising the peptidesor their derivatives as therapeutically effective ingredients areadministered parenterally, they were not tested for toxicity.

EXAMPLES

[0045] A better understanding of the present invention may be obtainedin light of the following examples which are set forth to illustrate,but are not to be construed to limit the present invention.

Example 1

[0046] Generation of Recombinant βig-h3 Fas-1 Domain Proteins and Assayfor Cell Attachment Activity

[0047] To find the amino acids essential to the cell adhesion of βig-h3,the ability of each of the four repeat fas-1 domains to mediate the celladhesion of βig-h3 was examined. To this end, four recombinant proteinsthat contained the four repeat fas-1 domains respectively were generatedfor cell attachment activity assay.

[0048] 1-1: Production of Recombinant βig-h3 fas-1 Proteins EncodingEach of Four Repeat Domains

[0049] Four DNA fragments encoding the 1^(st) to 4^(th) domainscorresponding to the bases 133 to 236, 242 to 372, 373 to 501, and 502to 632 of the βig-h3 gene, respectively, were generated by PCR andcloned into pET-29β (Novagen) to construct expression vectors for fas-1domains, named βig-h3 D-1, βig-h3 D-II, βig-h3 D-III, and βig-h3 D-IV,as shown in FIG. 1a. 6 tandem repeated histidine residues were providedto the C-termini of the DNA fragments to make a His-tag, which is usefulfor the purification of the expressed proteins by use of Ni-NTA resin(Quiagen). E. coli strain BS21 (DE3) was transformed with each of therecombinant expression vectors and cultured in LB media containing 50μg/ml kanamycin. The recombinant βig-h3 proteins were induced byculturing the transformants in the presence of 1 mM IPTG, followed bycentrifugation. The pellet was suspended in a lysis buffer consisting of50 mM Tris-HCl (pH 8.0), 100 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.5mM DTT and then sonicated to lyse the cells. After five repetitions ofthe procedure, centrifugation was conducted to separate a supernatantfrom which the proteins of interest were purified through a columnfilled with Ni-NTA resin.

[0050] On SDS-PAGE, the recombinant protein βig-h3 D-I was detected at14.4 Kda, while all of the recombinant proteins βig-h3 D-II, βig-h3D-III, and βig-h3 D-IV were detected at 21.5 KDa, as shown in FIG. 1b.

[0051] 1-2: Assay for the Cell Attachment Activity of Recombinant βig-h3fas-1 Domain Proteins

[0052] The recombinant βig-h3 fas-1 domain proteins prepared above wereassayed for cell attachment activity. First, the recombinant βig-h3proteins were let to adhere to the bottoms of 96-well microcultureplates (Falcon) by incubation at 37° C. for 1 hour and blocked with PBScontaining 0.2% BSA. HCE cells were suspended in culture media at adensity of 2×10⁵ cells/ml. 0.1 ml of the cell suspension was added toeach well of the plates coated with the recombinant proteins. Followingincubation at 37° C. for 1 hour, unattached cells were removed bywashing with PBS. Attached cells were incubated for 1 hour at 37° C. in50 mM citrate buffer, pH 5.0, containing 3.75 mM p-nitrophenol-N-acetyl1-β-D-glycosaminide as a hexosaminidase substrate and 0.25% TritonX-100, followed by the addition of 50 mM glycine buffer, pH 10.4,containing 5 mM EDTA to block the enzyme activity. A measurement wasmade of absorbance at 405 nm in a Multiskan MCC/340 microplate reader.Absorbance results are shown in FIG. 2.

[0053] As seen in the histogram of FIG. 2, HCE cells showed cellattachment and spreading activity comparable to the activity of wildtype βig-h3 (βig-h3-WT) in both plates coated with the recombinant2^(nd) fas-1 domain protein (βigh3-D-II) and recombinant 4^(th) fas-1domain protein (β igh3-D-IV) while exhibiting weak activity in the platecoated with the recombinant 1^(st) fas-1 domain protein (β igh3-D-I). Onthe other hand, almost no activity was detected in the plate coated withthe recombinant fas-1 domain III protein (β igh3-D-III).

[0054] Either the 2^(nd) or 4^(th) fas-1 domain is sufficient to mediatecell adhesion and spreading, indicating that amino acids essential tocell adhesion and spreading are present in each of the two domains.

[0055] 1-3: Identification of Receptors for βig-h3 fas-1 Domain Proteins

[0056] Using the recombinant βig-h3 2^(nd) and 4^(th) domain proteins,each showing cell attachment and spreading activity, βig-h3 receptorswere examined. In this regard, effects of function-blocking monoclonalantibodies against various integrin subunits on the adhesion of HCEcells to a surface coated with βig-h3 were examined.

[0057] In detail, HCE was preincubated in an incubation solution (3×10⁵cells/ml) in the presence of each of the monoclonal antibodies (5 μg/ml)against different types of integrins at 37° C. for 30 min. Thepreincubated cells were transferred onto plates precoated with βig-h3proteins and then incubated further at 37° C. for 1 hour, followed bythe quantitative analysis with hexosaminidase substrate as described inExample 1-2. The quantitative results are given in FIG. 3, in which thevalues are expressed as percentages of the number of cells adhering inthe absence of monoclonal antibodies.

[0058] As seen in FIG. 3, the 2^(nd) or 4^(th) fas-1 domain-mediatedcell adhesion was almost completely inhibited by both antibodies againstα3 and β1 integrin subunits. These results indicate that both the 2^(nd)and 4^(th) fas-1 domains are associated with α3β1 integrin to mediatethe cell attachment activity and have amino acids essential to themediation.

Example 2

[0059] Amino Acids, Aspartic Acid and Isoleucine, Essential to the CellAttachment and Spreading Activity of βig-h3

[0060] 2-1: Amino Acid Sequence Alignment of Various Proteins Containingfas-1 Domains

[0061] To identify amino acids responsible for cell adhesion from 2^(nd)and 4^(th) fas-1 domains of βig-h3, each having cell attachment andspreading activity, not only the internal repeat fas-1 domains ofβig-h3, but also other proteins containing fas-1 domains were subjectedto amino acid sequence alignment. As a result, two amino acids, that is,aspartic acid and isoleucine were found to be highly conserved at thesite near the H2 region of each fas-1 domain among various substrateproteins, as shown in FIG. 4. On the whole, both aspartic acid andisoleucine are conserved in various fas-1 domains, including the 2^(nd)and 4^(th) fas-1 domains of βig-h3, while only aspartic acid isconserved in the 1^(st) fas-1 domain. As for the 3^(rd) fas-1 domain, ithas neither of the two amino acids.

[0062] 2-2: Generation of Recombinant Proteins Containing SubstitutionMutants of the 4^(th) fas-1 Domain of βig-h3

[0063] In order to examine the essentiality of aspartic acid andisoleucine to the cell adhesion activity of βig-h3, mutants of therecombinant protein βig-h3 D-IV derived from the 4^(th) domain of βig-h3were generated in which serine, alanine, and serine were substituted forproline at position 616, aspartic acid at position 617, and isoleucineat position 618, respectively. The resulting mutated recombinantproteins were designated βig-h3 D-IV-sDI, βigh3 D-IV-PaI, βig-h3D-IV-PDs, and βig-h3 D-IV-sas (FIG. 5a). On SDS-PAGE, all of the mutatedrecombinant proteins were detected at the same position as that ofβig-h3 D-IV (FIG. 5b).

[0064] 2-3: Assay for Cell Attachment Activity of Recombinant ProteinsContaining Substitution Mutants of the 4^(th) fas-1 Domain of βig-h3

[0065] Examination was made of the cell attachment activity of themutated proteins wherein the Pro616, Asp617 and Ile618 of βigh3 D-IVwere substituted, in combination, with Ser, Ala and Ser, respectively,that is, β igh3 D-IV-sDI, β igh3 D-IV-PaI, β igh3 D-IV-PDs, and β igh3D-IV-sas. To this end, HCE cells were incubated at 37° C. for 1 hour inwell plates coated with the mutant proteins, followed by subjecting theattached cells to the hexosaminidase substrate as in Example 1-2. Theresults are given in FIG. 6.

[0066] As seen in FIG. 6, the mutant protein having Ala instead ofAsp617, named D617A (β igh3 D-IV-PaI) and the mutant protein having Serinstead of Ile618, named I618S (β igh3 D-IV-PDs) significantly blockedcell adhesion whereas the mutant protein having Ser instead of Pro616,named P616S (β igh3 D-IV-sDI) was found to show cell attachment activitycomparable to that of the wild type control. As for the mutant proteinin which the three amino acids were mutated, named P616S/D617A/I618S (βigh3 DIV-sas), its cell attachment activity was also far lower than thecontrol.

[0067] The loss of the 1^(st) fas-1 domain-mediated cell attachmentactivity in the 1^(st) fas-1 domain mutated at Asp617 or Ile618indicates that the aspartic acid at position 617 and isoleucine atposition 618 are very important to mediate cell attachment activity ofβig-h3.

[0068] These results agree with those of Example 1. Reviewing theresults of Example 1, the aspartic acid and isoleucine, which areidentified to be essential to mediate cell attachment activity, areconserved in both the 2^(nd) and 4^(th) fas-1 domains showing cellattachment activity, while only the aspartic acid is conserved in the1^(st) fas-1 domain, which shows weak cell attachment activity. On theother hand, the 3^(rd) domain with no cell attachment activity hasneither of the two amino acids. Consequently, the results taken togetherdemonstrate that Asp617 and Ile618 are very important to mediate cellattachment and spreading activity of βig-h3.

EXAMPLE 3

[0069] βig-h3-Mediated Synthetic Peptides with Cell Attachment,Spreading and Detachment Activity

[0070] With the confirmation of the indispensability of aspartic acidand isoleucine for the cell attachment activity, peptides containing thetwo amino acids were synthesized and assayed for cell attachment anddetachment activity.

[0071] 3-1: Synthesis of βig-h3 fas-1 Domain-Derived Proteins

[0072] Peptides derived from the 1^(st,) 2^(nd) and 4^(th) fas-1 domainsof βig-h3 and a control peptide were synthesized with the aid of anautomated multiple peptide synthesizer (PE/ABD 433) by standard solidphase procedures and they were isolated and purified by reverse phasehigh performance liquid chromatography. The synthesized peptides weredesigned to have KADHH (a.a 219-223) of SEQ. ID. NO. 1, NKDIL (a.a.354-358) of SEQ. ID. NO. 2, and EPDIM (a.a. 615-619) of SEQ. ID. NO. 3,corresponding to conserved sequences of the 1^(st,) 2^(nd) and 4^(th)fas-1 domains of βig-h3, respectively, and the control peptide DEMPI hadthe same amino acid composition as the peptide EPDIM, but was changed inits amino acid sequence, as shown in FIG. 7.

[0073] 3-2: Assay for Cell Attachment and Detachment Activity ofSynthetic Peptides

[0074] Effect of the peptides containing conserved sequences of fas-1domains of βig-h3 on βig-h3-mediated cell adhesion was examined with HCEcells. Cells were preincubated for 30 min in media containing 100 μM ofeach of the four synthetic peptides or no peptides and transferred toplastic culture dishes coated with 10 μg/ml BSA or 10 μg/ml βig-h3. Theattached cells were subjected to quantitative hexosaminidase analysis asin Example 1-2. The results are given in FIG. 8a.

[0075] As shown in FIG. 8a, the synthetic peptides NKDIL and EPDIM, eachcontaining both of the conversed aspartic acid-isoleucine residues,significantly blocked the HCE cell adhesion to βig-h3, showing excellentcell detachment activity, while the synthetic peptide KADHH containingonly the conserved aspartic acid weakly inhibited the cell adhesion. Onthe other hand, the control peptide DEMPI containing neither of theconversed amino acid residues did not affect cell adhesion and thus hadlow cell detachment activity.

[0076] The HCE cell adhesion to the recombinant proteins βigh3 D-II andβigh3 D-IV was also examined in the presence or absence of the syntheticpeptides. To this end, HCE cells were preincubated for 30 min in mediacontaining 100 μM of each of the four synthetic peptides or no peptidesand then transferred to plastic culture dishes coated with 10 μg/mlβigh3 D-II or D-IV. Following incubation, the attached cells werequantitatively analyzed with hexosaminidase as in Example 1-2. Theresults are shown in FIG. 8b.

[0077] As seen in FIG. 8b, blocking effects of the synthetic peptidesobtained when the 2^(nd) and 4^(th) fas-1 domains were used assubstrates were similar to those obtained when βig-h3 was used as asubstrate.

[0078] 3-3: Cell Adhesion of HCE Cells to Surfaces Coated with SyntheticPeptides

[0079] An experiment was carried out to determine whether each of thesynthetic peptides is able to mediate cell adhesion. Well plates werecoated with each peptide at concentrations of 0, 20, 40, 60, 80, 100 and120 μM and then incubated with HCE cells at 37° C. for 1 hour. Theattached cells were quantitatively analyzed with hexosaminidase as inExample 1-2. The results are shown in FIG. 9a.

[0080] The synthetic peptides NKDIL and EPDIM, each containing bothaspartic acid and isoleucine, were found to mediate cell adhesion indose-dependent manners. The synthetic peptide KADHH was also capable ofmediating cell adhesion in a dose-dependent manner, but was low inactivity compared to NKDIL and EPDIM. As expected, the control peptidehaving neither of the two amino acids was not active in cell adhesion.

[0081] 3-4: Identification of Receptors for Mediating Cell Attachment,Spreading and Detachment Activity of Synthetic Peptides

[0082] Function-blocking experiments using monoclonal antibodies againstvarious integrin subunits were carried out to examine whether α3β1integrin, known as a surface receptor of βig-h3, would be involved inthe cell adhesion mediated by the synthetic peptides EPDIM and NKDIL.HCE was preincubated at 37° C. for 30 min in the presence of each of themonoclonal antibodies (5 μg/ml) against different types of integrins.The preincubated cells were transferred onto plates precoated with 100μM EPDIM or 100 μM NKDIL and then incubated further at 37° C. for 1hour, followed by the quantitative analysis with hexosaminidasesubstrate as in Example 1-2. The quantitative results are given in FIG.9b.

[0083] As seen in FIG. 3, the 2^(nd) or 4^(th) fas-1 domain-mediatedcell adhesion was almost completely inhibited by both antibodies againstα3 and β1 integrin subunits. These results indicate that both the 2^(nd)and 4^(th) fas-1 domains are associated with α3β1 integrin to mediatecell adhesion.

[0084] 3-5: Effect of Synthetic Peptides on Cell Adhesion to VariousExtracellular Matrix Peptides

[0085] To determine whether the inhibitory activity of syntheticpeptides EPDIM and NKDIL against cell adhesion is specific to βig-h3,effects of the synthetic peptides on cell adhesion to various matrixproteins were examined. HCE cells were preincubated in media added with100 μM of the synthetic peptide EPDIM or NKDIL or none of them and thenapplied to plates coated with various matrix proteins, including βig-h3,fibronectin, laminin, vitronectin, type I and type II collagen. After 1hour of incubation, the attached cells were quantitatively analyzed withhexosaminidase as in Example 1-2. The results are shown in FIG. 10a.

[0086] As shown in FIG. 10a, the synthetic peptides NKDIL and EPDIMefficiently blocked cell adhesion not only to βig-h3 but also laminin,showing excellent cell detachment activity. However, they weaklyinhibited cell adhesion to fibronectin and did not affect cell adhesionto type I and type II collagen and vitronectin at all. Accordingly, theresults, taken together, suggest that the synthetic peptides NKDIL andEPDIM specifically compete with α3β1 integrin-interacting molecules.

[0087] Cell adhesion to βig-h3, laminin and fibronectin, which was foundto be blocked by the synthetic peptides NKDIL and EPDIM, was examined atvarious concentrations of EPDIM. HCE cells were incubated in mediacontaining the synthetic peptide EPDIM at concentrations of 0, 200, 400,600, 800, 1,000 and 1,200 μM and applied to plates coated with βig-h3,fibronectin and laminin. Following incubation for 1 hour, the attachedcells were quantitatively analyzed with hexosaminidase as in Example1-2. The results are shown in FIG. 10b.

[0088] As seen in the curves, the cell adhesion to βig-h3, fibronectinand laminin became weak as the concentration of the synthetic peptideEPDIM increased. That is, the inhibitory effects of EPDIM on celladhesion to βig-h3, fibronectin and laminin were dose-dependent.

[0089] In the present invention, as described above, there are preparedthe synthetic peptides NKDIL and EPDIM, both containing aspartic acidand isoleucine essential for cell attachment and spreading activity intheir conserved motifs. The peptides contain conserved sequences of the2^(nd) and 4^(th) fas-1 domains of βig-h3, each domain found to showcell attachment activity, and they induce cell adhesion through α3β1integrin, known as a functional cell receptor. In the case of variousextracellular matrix proteins known to bind to α3β1 integrin, such asthrombospondin, laminin, collagen type IV, etc., neither characteristicsequences having homology among their active sites, nor characteristicconserved binding motifs for α3β1 integrin have been discovered, thusfar. However, given that the synthetic peptides NKDIL and EPDIM, eachcontaining both the conserved amino acids aspartic acid-isoleucine, bindspecifically to α3β1 integrin so as to mediate cell attachment activity,the conserved aspartic acid-isoleucine is identified as thecharacteristic conserved binding motif for α3β1 integrin in accordancewith the present invention. Therefore, the peptides of the presentinvention can be usefully used for studying cell attachment activitymediated through various extracellular matrix proteins, includingβig-h3, and for developing cell attachment, spreading anddetachment-promoting peptides.

INDUSTRIAL APPLICABILITY

[0090] In accordance with the present invention, it is disclosed that,among four fas-1 domains of βig-h3, which is a cell adhesion molecule,the 2^(nd) and 4^(th) domains show cell attachment and detachmentactivity, and each have a conserved aspartic acid-isoleucine sequence,essential for the cell attachment and detachment activity. Given thesefindings, there are prepared peptides NKDIL and EPDIM, which contain thefunctional amino acid residues and induce cell adhesion through α3β1integrin. The peptides of the present invention can be usefully used forstudying cell attachment activity mediated through various extracellularmatrix proteins, including βig-h3, and for developing cell attachment,spreading and detachment-promoting peptides.

[0091] The present invention has been described in an illustrativemanner, and it is to be understood that the terminology used is intendedto be in the nature of description rather than of limitation. Manymodifications and variations of the present invention are possible inlight of the above teachings. Therefore, it is to be understood thatwithin the scope of the appended claims, the invention may be practicedotherwise than as specifically described.

1 4 1 5 PRT Homo sapiens 1 Lys Ala Asp His His 1 5 2 5 PRT Homo sapiens2 Asn Lys Asp Ile Leu 1 5 3 5 PRT Homo sapiens 3 Glu Pro Asp Ile Met 1 54 5 PRT Homo sapiens 4 Asp Glu Met Pro Ile 1 5

What is claimed is:
 1. A peptide and its derivatives having cellattachment, spreading and detachment activity, comprising an amino acidsequence represented by XXDIX.
 2. The peptide and its derivativesaccording to claim 1, wherein the peptide and its derivatives have anamino acid sequence consisting of at least five amino acid residuesrepresented by XXDIX, comprising apartic acid and iloleucine as aconserved sequence.
 3. The peptide and its derivatives according toclaim 2, wherein the amino acid sequence is represented by NKDIL of SEQ.ID. NO.
 2. 4. The peptide and its derivatives according to claim 2,wherein the amino acid sequence is represented by EPDIM of SEQ. ID. NO.3.
 5. The peptide and its derivatives according to claim 1, wherein thepeptide and its derivatives mediate cell attachment and spreadingactivity through α3β1 integrin.
 6. The peptide and its derivativesaccording to claim 1, wherein the peptide and its derivatives showinhibitory activity against cell adhesion to βig-h3, fibronectin orlaminin.
 7. Pharmaceutical compositions, comprising the peptide or itsderivatives of claim 1 as a pharmaceutically active ingredient.
 8. ThePharmaceutical compositions according to claim 7, wherein thepharmaceutical compositions are therapeutically effective for woundhealing, tissue regeneration, or cancer metastasis resistance improvingbiointerface of biomaterials and tissue implants.